Bidirectional promoters in nannochloropsis

ABSTRACT

Exemplary embodiments provided herein include novel promoters isolated from the microalgae,  Nannochloropsis . These promoters drive gene expression in a bidirectional manner, and are especially useful for the genetic manipulation of  Nannochloropsis  and other organisms. The inventors herein successfully used these promoters (in both parallel and antiparallel orientations with respect to a Sh ble gene cassette) to impart zeocine-resistance to  Nannochloropsis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This continuation application claims the benefit and priority of U.S.Non-Provisional patent application Ser. No. 12/706,683 filed Feb. 16,2010, titled “Bidirectional Promoter in Nannochloropsis,” which claimsthe benefit and priority of U.S. Provisional Patent Application Ser. No.61/207,564 filed on Feb. 13, 2009, titled “Bidirectional Promoter inNannochloropsis,” all of which are incorporated by reference herein.

This continuation application is related to U.S. Non-Provisional patentapplication Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-BasedVectors for Algal Cell Transformation,” which in turn claims the benefitand priority of U.S. Provisional Patent Application Ser. No. 61/059,672filed on Jun. 6, 2008, titled “VCP-Based Vector For NannochloropsisTransformation,” the disclosures of both which are incorporated byreference herein.

REFERENCE TO SEQUENCE LISTINGS

The present application is filed with sequence listing(s) attachedhereto and incorporated by reference.

FIELD OF THE INVENTION

This invention relates to molecular biology, and more specifically tothe transformation of algal cells and the expression of exogenousdeoxyribonucleic acid (DNA).

SUMMARY OF THE INVENTION

Exemplary embodiments provided herein include novel promoters isolatedfrom the microalgae, Nannochloropsis. These promoters drive geneexpression in a bidirectional manner, and are especially useful for thegenetic manipulation of Nannochloropsis and other organisms. Theinventors herein successfully used these promoters (in both parallel andantiparallel orientations with respect to a Sh ble gene cassette) toimpart zeocine-resistance to Nannochloropsis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows exemplary transformation construct NT6.

FIG. 1B shows exemplary transformation construct NT7.

FIG. 2 shows a table reflecting the exemplary results of a growth assayused to analyze the transformants that resulted from the threetransformation constructs, NT6, NT7, and PL90.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary embodiments provided herein include novel promoters isolatedfrom the microalgae, Nannochloropsis. These promoters drive geneexpression in a bidirectional manner, and are especially useful for thegenetic manipulation of Nannochloropsis and other organisms. Theinventors herein successfully used these promoters (in both parallel andantiparallel orientations with respect to a Sh ble gene cassette) toimpart zeocine-resistance to Nannochloropsis.

FIGS. 1A-1B show two exemplary transformation constructs, transformationconstruct NT6 (FIG. 1A) and transformation construct NT7 (FIG. 1B).Transformation construct NT6 as shown in FIG. 1A includes abidirectional promoter sequence 110 (SEQ. ID. NO. 1), a Sh ble genecassette sequence 120, and a 3′ untranslated region sequence (UTR) 130.Transformation construct NT7 as shown in FIG. 1B includes abidirectional promoter sequence 110 (SEQ. ID. NO. 2), a Sh ble genecassette sequence 120, and a 3′ untranslated region sequence (UTR) 130.

When analyzing a Nannochloropsis genomic sequence, the inventors foundtwo divergently transcribed Vcp genes orientated back to back (i.e.transcription must have been initiated from the nucleotide sequenceseparating both genes) separated by several hundred nucleotides. Theinventors believed that this nucleotide sequence that separated thedivergently transcribed Vcp genes included the requisite regulatoryelements to drive expression of both divergently transcribed Vcp genes.

The inventors created transformation constructs NT6 and NT7 to confirmthey had discovered a bidirectional promoter in the Nannochloropsisgenome. The bidirectional promoter was amplified from theNannochloropsis genome using Polymerase Chain Reaction (PCR) and otherstandard techniques. A Nannochloropsis transformation construct (orvector) was constructed using a pJet vector as the backbone. Thebidirectional promoter was cloned in both a parallel (NT6) and ananti-parallel (NT7) fashion relative to a standard zeocine-resistance(Sh ble) cassette. A Vcp 3′-UTR was placed immediately downstream of thezeocine-resistance (Sh ble) cassette in both constructs.

The NT6 and NT7 transformation constructs were cut out via restrictionenzyme digestion and the transformation construct purified after DNA gelelectrophoreses. A PL90 transformation construct, as described in U.S.Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8,2009, titled “VCP-Based Vectors for Algal Cell Transformation,” thatincluded another Vcp-promoter was linearized to be used as a comparisonto the NT6 and NT7 transformation constructs. All three transformationconstructs in equimolar amounts were used to transform Nannochloropsiscells, which were then allowed to incubate at room temperature under ˜85μE light on solid selective media (with a zeocine concentration of 2micrograms per milliliter) for several weeks until visible colonies wereformed.

FIG. 2 shows a table reflecting the exemplary results of a growth assayused to analyze the transformants that resulted from the threetransformation constructs, NT6 (FIG. 1A), NT7 (FIG. 1B), and PL90. Theinventors found that the bidirectional promoter sequence 110 (FIGS.1A-1B) in both orientations (i.e. parallel and anti-parallel) withineach transformation construct (i.e. NT6 and NT7) drove much higherlevels of gene expression than the Vcp-promoter used in the PL90transformation construct.

Referring again to FIG. 2, the growth assay utilized zeocine, anantibiotic that kills most aerobic cells by binding and cleaving the DNAin the aerobic cells. The Sh ble gene product produced by the Sh blegene cassette sequence 120 (FIGS. 1A-1B) prevents toxicity by binding tozeocine and inactivating it. Accordingly, a higher level of Shble-transgene expression in an algal cell will lead to a greater abilityfor the algal cell to survive in higher zeocine concentrations.

Zeocine was utilized in a kill-curve as follows:

1. 1 mL aliquots of F2 media were added to wells in a 24-well plate.

2. Zeocine was added to the wells with F2 media to achieve finalconcentrations of zeocine as listed in the left-hand column of the FIG.2 table (i.e. the final concentrations of zeocine ranged from 0 ug/ml to200 ug/ml).

3. Colonies were randomly picked from agar plates containing the NT6,NT7, and PL90 transformants (the number of transformants tested for eachtransformation construct is given as the final line-item in the FIG. 2table).

4. Each colony was resuspended in 30 uL of N2 media.

5. 2 uL of each colony-resuspension was added to the wells containingthe increasing amounts of zeocine (wells without zeocine were includedas controls, but are not shown in the FIG. 2 table).

6. The 24-well plates were allowed to incubate under 85 uE light for 1week.

7. Optical density measurements (at 750 nm) for each well were obtainedwith a spectrophotometer.

8. For each transformant, the highest zeocine concentration wasdetermined required to enable at least 50% growth of the cell line ascompared to the no-zeocine controls.

The exemplary data reflected in the FIG. 2 table shows the number ofcolonies that had at least 50% survival up to the specified zeocineconcentration. The data in the FIG. 2 table shows that the NT6 and NT7transformation constructs withstood higher levels of zeocine than didthe PL90 transformation construct. Because the promoter was the onlyvariable between the NT6/NT7 and PL90 transformation constructs, itfollowed that the increased survival rate of the NT6/NT7 transformationconstructs was due to the bidirectional promoter, which is apparentlystronger than the Vcp-promoter used in the PL90 transformationconstruct. It should be noted that wild-type Nannochloropsis willgenerally not survive zeocine concentrations of 2 micrograms permilliliter and higher.

The various exemplary bidirectional promoters provided herein have beenused to drive expression of genes introduced to Nannochloropsis viatransformation. They also may be used to perform activation-taggingrandom insertional mutagenesis experiments. To achieve certainphenotypes through genetic manipulation, up-regulation of the expressionof certain genes (as opposed to the down-regulation or the knocking outof certain genes) may be required. Forward genetics may be performedwith the bidirectional promoter in a highly efficiently manner becausethe promoter can activate genes in both directions. A typical activationtagging experiment, to achieve higher oil production, could be performedas follows: A transformation construct comprising the bidirectionalpromoter as depicted in FIG. 1A or 1B, a selection gene (e.g. the Sh blegene), and an 5′ UTR, is isolated via PCR or restriction digest of theplasmid containing the bidirectional promoter would be introduced intoNannochloropsis via previously-described methods. The bidirectionalpromoter would each be inserted into the Nannochloropsis genome inrandom locations when individual transformants are analyzed, and at somefrequency, it would insert upstream of the genes involved in, e.g.,lipid biosynthesis. Compared to the promoters of genes involved withlipid biosynthesis, the Vcp promoters, being strong promoters, wouldlikely drive higher expression of the gene(s). An assay, such as theNile Red assay, could be performed to identify transformants thatproduce more lipids.

The various exemplary bidirectional promoter sequences provided hereinmay be used to perform RNA-interference (RNAi). RNAi is based on thepresence of dsRNA, either introduced exogenously or produced within acell itself. The bidirectional promoter provides for a facile system toperform RNAi, as the gene of interest can be expressed in parallel andanti-parallel fashions, thus making reverse complements of one another(dsRNA).

What is claimed is:
 1. A bidirectional promoter for a transformationconstruct for algal cell transformation, the bidirectional promotercomprising the nucleotide sequence of SEQ. ID. NO.
 2. 2. Thebidirectional promoter of claim 1, wherein the algal cell is of algalgenus Nannochloropsis.
 3. The bidirectional promoter of claim 1, whereinthe bidirectional promoter promotes transcription of a first nucleotidesequence adjacent to a first side of the bidirectional promoter in a 3′direction and the bidirectional promotes transcription of a secondnucleotide sequence adjacent to a second side of the bidirectionalpromoter in a 5′ direction.
 4. The bidirectional promoter of claim 3,wherein the bidirectional promoter promotes transcription of the firstand the second nucleotide sequences at a same time.
 5. The bidirectionalpromoter of claim 3, wherein the first nucleotide sequence includes aselection marker gene.
 6. The bidirectional promoter of claim 3, whereinthe second nucleotide sequence includes a selection marker gene.
 7. Thebidirectional promoter of claim 5 or claim 6, wherein the selectionmarker gene is a Sh Ble gene.